結構性基因及非結構性基因報導質體的建構 - 探討登革熱二型病毒PL046之質體建構及報導基因的選擇

在結構性基因報導質體建構過程中，針對pcDNA3-NCS/5’S3’UTR 進行基因定序。發現對應於登革熱病毒NGC strain 基因序列的 2512 位置（第 806 個胺基酸位置，位於 NS1 蛋白）發生了一個點突變，使得原本應為榖胺醯胺（glutamine）的 CAA 變成 TAA，造成蛋白質轉譯的停止（請參照結果 4.1.7）。由於此胺基酸位於非結構性基因 NS1 內，不影響結構性基因的轉譯，所以接續的結構性報導質體的建構仍以此段序列為結構性基因來源。

由於 pcDNA3-NCS/5’S3’UTR 的結構性基因來源是 pcDNA3-NCS/

DV2F（P1），在定序後發現在非結構性基因上有一無義突變（nonsense mutation），故接續的非結構性質體建構的基因來源改以 TA cloning 所得到含有登革熱病毒基因序列的 pCR-XL-TOPO/DV2F（S）當作非結構性基因的來源。

5.5 結構性基因報導質體及非結構性基因報導質體的表現

過去有文獻指出，在bicistronic 的狀況下，冷光基因（luciferase）位於第一個ORF 的表現量較位於第二個 ORF 且前方加入 EMCV IRES 的表現量約多5 倍(Hennecke et al., 2001)。結果 4.6 顯示，在 bicistronic 的狀況下，當

報導基因位於第一個（非結構性基因報導質體）或第二個 ORF（結構性基因報導質體）時，產生出來的藍色細胞數可以說差異十分懸殊，即使在第二個 ORF 前方加入了 EMCV IRES，雖然產生的藍色細胞數有增加，但是與位於第一個ORF 的報導基因比起來，產生的藍色細胞數仍然只有二十分之一，而且顏色的深淺，也有明顯的差異。雖然上述Hennecke 等人的研究結果顯示，利用 EMCV IRES 進行轉譯的效果約為利用 cap dependent transaltion 的 5 分之 1，但由於報導基因並不相同，所以產生的結果可能有所差異。

建構結構性基因及非結構性基因報導的目的是要進行登革熱病毒組裝訊號的分析。整個策略是將結構性基因報導質體或非結構性基因報導質體轉染至細胞後，讓細胞合成出帶有報導基因及登革熱病毒基因（結構性或非結構性基因）序列的 RNA，同時，也對細胞進行登革熱病毒的感染，以產生登革熱病毒所需的蛋白。在登革熱病毒進行組裝時，會將具有組裝訊號的RNA 進行包覆，並釋放至細胞外。將產生出的病毒收集後，再感染新的 BHK-21 細胞，如果能在被感染的細胞內偵測到報導基因的表現（藍色細胞的生成），即可得知報導基因上帶有的登革熱病毒序列具有病毒組裝訊號。

在這種實驗架構下，報導基因的表現量及活性一定要足夠。因為帶有報導基因及登革熱病毒基因（結構性或非結構性基因）序列的RNA 最後進入細胞內的數量應該不多，而且可能無法複製；在這種狀況下，轉譯出的 β-半乳糖苷酶是否足夠讓細胞轉成藍色，是值得關注的。尤其當報導基因位於第二個 ORF 時，表現的狀況原本就不佳，如果 RNA 經過病毒組裝，

再經由感染進入細胞，產生的β-半乳糖苷酶必定更少。未來在進行組裝訊號分析時，可能要針對這個問題，尋找其他的報導基因，例如螢光或冷光基因，嘗試改善偵測狀況；或者，改用RT-PCR 的方式，藉由偵測報導基因 RNA 序列的存在與否，觀察 RNA 是否被病毒進行組裝。

陸、結論

研究結果顯示，所獲得的登革熱病毒全長 cDNA clone 置於含有 enhancer 的 CMV 啟動子下游時，並無法表現出具有感染性的登革熱病毒，

可能是由於登革熱病毒所產生的蛋白對 E. coli 有毒性，且 CMV 啟動子在 E. coli 產生 leaky，造成得到的質體都是經過突變不具感染性的質體。另外，

亦有可能是由於質體建構時，未去除 5’及 3’端的非病毒核苷酸序列，導致病毒RNA 無法被複製，因此無法偵測到任何具有感染性的病毒顆粒生成。

在X-gal staining分析含有病毒基因的報導質體表現時發現，當報導基因與病毒基因分別使用各自的ORF（bicistronic）的狀況下，報導基因位於在上游的ORF時，產生的藍色細胞數最多，在 3.5 公分的培養皿上約可生成 1.6×10⁵ 個藍色細胞；當報導基因位於下游的ORF時，在前方加入IRES的報導基因所產生的藍色細胞數較未加入IRES的多，但藍色細胞數分別只有在位於上游ORF的二十分之一及二千分之一。若報導基因與病毒基因共用同一個ORF 時（ monocistronic ），產生的藍色細胞約只有位於上游 ORF

（bicistronic）的五百分之一。未來在進行組裝訊號分析時，建議採用 bicistronic的方式，並使用其它的報導基因，例如螢光或冷光基因，以嘗試改善當報導基因位於下游ORF不易被偵測的狀況。或者，改用RT-PCR的方式，藉由偵測報導基因RNA序列的存在與否，觀察RNA是否被病毒進行組裝。

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pcDNA3 5446 bp

Neomycin Ampicillin

CMV promoter T7 promoter

pBluescript Ⅱ SK(+) 2961 bp

MCS

Ampicillin T7 promoter

lac promoter HindIII (890)

XbaI (984) HincII (2286)

XbaI (2227)

pc NCS

54

DNA3-11 bp

MCS

Neomy in c Ampicillin CMV promoter

T7 promoter XbaI (949)

pcDNA3 : pBluescriptⅡSK(+):

d III (Klenow) + Xba I digestion HincⅡ+ Xba I digestion

圖一、Constructio of

pcDNA3-NCS: New cloning site from pBluescriptⅡSK(+) Hin

n NCS

pCR-XL- /DV2F 14 p

TOPO 256 b

CDS of DV2

Full length cDNA of DV2 genome Zeocin

Kanamycin

lac promoter 3'UTR

5'UTR

ClaI (110 ) 68

XbaI (3 1)4

XbaI (11126)

pCR-XL-TOPO: TA cloning vector RT-PCR product

、TA cloning – p

CDS: Coding sequence

DV2: Dengue virus 2 PL046 strain F: Full length cDNA

CR-XL-TOPO/DV2F 圖二

pcDNA3-NCS

Full length cDNA of DV2 genome Zeocin

pcDN A3-NCS /DV2F 16089 bp

CDS of DV2

Full length cDNA of DV2 genome Ampicillin

圖三、Construction of pcDNA3-NCS/DV2F CDS: Coding sequence

DV2: Dengue virus 2 PL046 strain F: Full length cDNA

NCS: New cloning site

Cla I + Xba I digestion

627

圖四、單一ORF 非結構性基因報導質體建構示意圖

5’: 5’UTR DV2: Dengue virus 2 PL046 strain

NCS: New cloning site NS: Nonstructural genes of DV2 from P1 Z: lacZ (YEP) w/o Kozak sequence MONO: monocistronic

pcDNA3-NCS/3'UT R 5856 bp

Ampicillin

Neomycin CMV promoter

T7 promoter 3'UTR

Xba I (139 4) BamHI 937) (

pcDNA 3 -NCS: PCR product of 3’UTR:

BamH I + Xba I digestion BamH I + Xba

圖五、Construction of pcDNA3-NCS/3’UTR DV2: Dengue virus 2 PL046 strain NCS: New cloning site

I digestion

pcDNA3-NCS/5'3'UT R 5955 bp

Ampicillin

Neomycin CMV promoter

T7 promoter

3'UTR 5'UTR ClaI (902)

HindIII (1006)

pcDNA3 -NCS/3’UTR: PCR product of 5’UTR:

圖六、Construction of pcDNA3-NCS/5’3’UTR

DV2: Dengue virus 2 PL046 strain

Cla I + HindⅢdigestion Cla I + HindⅢdigestion

5’: 5’UTR

NCS: New cloning site

pcDNA3-NCS/5'Z(YEP)3'UTR 8985 bp

Ampicillin lin

Neomycin

CMV promoter T7 promoter

lacZ

3'UTR 3'UTR

5'UTR

HindIII (1006) dIII (1006)

pcDNA3-NCS/ 'Z(YEP)3'UTR 8985 bp

5 Ampicil

Neomycin

CMV prom ter o T7 promoter

lacZ 5'UTR

Hin

BamHI (4066)

HindⅢ+ BamH I digestion HindⅢ+BamH I digestion

圖七、Construction of pcDNA3-NCS/5’Z(YEP)3’UTR

NCS: New cloning site

Z(YEP): lacZ from YEP363 w/o Kozak sequence 5’3’UTR/pcDNA3-NCS: PCR product of lacZ:

5’: 5’UTR

pcDNA3-NCS/5’Z3’UTR: pcDNA3-NCS/DV2F BamH I + Xba I digestion BamH I + Xba I digestion

圖八、construction of pcDNA3-NCS/5’ZNS(MONO)3’UTR

5’: 5’UTR S: Coding sequence DV2: Dengue virus

NCS: New cloning site Z: lacZ(YEP) w/o Kozak sequence NS: Nonstructural genes of DV2 from P1

2 PL046 strain F: Full length cDNA

5’: 5’UT

NCS: New cloning si

Z(YEP): 3

圖九、報導基因的選擇–來源及轉譯起始點之比較 R K: Kozak sequence

te Z(BAG): lacZ from BAG lacZ from YEP36

pcDNA3-NCS/5'KZ(YEP)3'UTR 8988 bp

Ampici n lli

Neomycin

CMV prom ter o T7 promoter

lacZ (Kozak)

3'UTR

5'UTR

BamHI (4069)

5’3’UTR/pcDNA3-NCS: PCR product of lacZ:

EcoR V + BamH I digestion Sma I + BamH I digestion

5’: 5’UTR

K: Kozak sequenc NCS: New cloning site

Z(YEP): lacZ from YEP363 with Kozak sequence 圖十、Construction of pcDNA3-NCS/5’KZ(YEP)3’UTR

pcDNA3-NCS/5'KZ(BAG)3'UTR 8988 bp

Ampicillin

Neomycin

CMV promoter T7 promoter

lacZ (Kozak)

3'UTR

5'UTR

BamHI (4069)

圖十一、Construction of pcDNA3 G)3’UTR

quence ite

with Kozak sequence 5’3’UTR/pcDNA3-NCS: PCR product of lacZ:

Eco RV + BamH I digestion Sma I + BamH I digestion

-NCS/5’KZ(BA 5’: 5’UTR

K: Kozak se

NCS: New cloning s Z(BAG): lacZ from BAG

pGEM-T/lacZ

6089 bp

Ampicillin

T7 prom tero

SP6 promo rte

lacZ

AscI (3125) PacI (58)

PmeI (3135)

NotI (63)

NotI (3152)

lacZ from BAG

pGEM-T vector PCR product of lacZ

圖十二、TA cloning – pGEM-T/lacZ lacZ:

Dengue virus 2 PL046 strain : Internal ribosomal entry site tructural genes of DV2 from P1

lacZ(BAG) with Kozak sequence

圖十三、結構性基因報導質體建構示意圖

5’: 5’UTR DV2:

EMCV: Encephalomyocarditis virus IRES NCS: New cloning site S: S

Sp: Spacer DNA Z:

＊: nonsense mutation in NS1 gene

圖十四、Construction of pcDNA3-NCS/5’S3’UTR 5’: 5’UTR

DV2: Dengue virus 2 PL046 strain F: Full length cDNA

NCS: New cloning site

Structural genes of DV2 from P1

pcDNA3-NCS/5’3’UTR: pcDNA3-NCS/DV2F:

HindⅢ(Klenow) + Cla I PpuM I (Klenow) + Cla I

pcDNA3-NCS/5'SZ3'UTR 12264 bp

Structural gene of DV2

Neomycin

Ampicillin CMV promoter

T7 prom er ot

lacZ 3'UTR

5'UTR

lenow)

圖十五、Construction of pcDNA3-NCS/5’SZ3’UTR 5’: 5’UTR

ue virus 2 PL046 strain DV2 from P1

k sequence pcDNA3-NCS/5’S3’UTR: pGEM-T/lacZ:

EcoR V digestion Sac II、SpeI digestion(K

DV2: Deng

NCS: New cloning site S: Structural genes of Z: lacZ(BAG) with Koza

pGEM-T/Spacer 7905 bp

T7 promoter

SP6 prom er ot

Insert

lacZ

Using for spacer DNA (nt1101-4727)

R V digestion cⅡdigestion

圖十

pGEM-T/lacZ: pGEM-T/lacZ:

Eco Hin

六、Construction of pGEM-T/spacer

pcDNA3-NCS/5’SZ3’UTR: PCR product of Spacer:

stion

圖十七、

Spac cer

5’: 5’UTR DV2: Dengue virus 2 PL046 strain NCS: New cloning site S: S ructural genes of DV2 from P1 Sp: Spacer DNA Z: lacZ (BAG) with Kozak sequence Not I + Pac I digestion Not I + Pac I dige

Construction of pcDNA3-NCS/5’SZSp3’UTR er DNA: nt 1101 ~ nt 4727 of pGEM-T/ Spa

pcDNA3.0-NCS/5’SZSp3’UTR PCR product of EMCV IRES:

圖十八、Construction of pcDNA3.0-NCS/5’S(IRES)ZSp3’UTR 5’: 5’UTR

EMCV: Encep ry site

NCS: New clo rom P1

Sp: Spacer DNA Z: lacZ(BAG) with Kozak sequence Pme I + Asc I digestion Pme I + Asc I digestion

DV2: Dengue virus 2 PL046 strain halomyocarditis virus IRES: Internal ribosomal ent

ning site S: Structural genes of DV2 f

圖十九、非結構性基因報導質體建構示意圖

’: 5’UTR DV2: Dengue virus 2 PL046 strain MCV: Encephalomyocarditis virus IRES: Internal ribosomal entry site

CS: New cloning site NS: N S

Z: lacZ (BAG) with Kozak sequence

onstructural genes of DV2 from 5

E N

Ampicillin

圖二十、Construction of pcDNA3.0-NCS/5’Z3’UTR 5’: 5’UTR

NCS: New cloning site

Z: lacZ (BAG) with Kozak sequence

peI digestion(Klenow) :

pcDNA3-NCS/5'Z3'UTR 9057 bp

Ampicillin

Neomycin

CMV promoter T7 promoter

lacZ

3'UTR

5' TRU

XbaI (4595) BamHI (4138)

V2F (S)

圖

5’: 5’UTR CDS: Coding sequence DV2: Dengue virus 2 length cDNA

NCS: New cloning si ozak sequence NS: Nonstructural genes of DV2 from S

pcDNA3-NCS/5’Z3’UTR: pCR-XL-TOPO/D

BamH I + Xba I digestion BamH I + Xba I digestion

二十一、Construction of pcDNA3-NCS/5’ZNS3’UTR

PL046 strain F: Full

te Z: lacZ (BAG) with K

ES:

圖二 R

PL046 strain arditis virus

(BAG) with Kozak sequence NS: Nonstructural genes of DV2 from S

pcDNA3-NCS/5’ZNS3’UTR: PCR product of EMCV IR Not I + BamH I digestion Not I + BamH I digestion

十二、Construction of pcDNA3-NCS/5’Z(IRES)NS3’UT

5’: 5’UTR DV2: Dengue virus 2

EMCV: Encephalomyoc IRES: Internal ribosomal entry site NCS: New cloning site Z: lacZ

a

2.5kb3kb4kb5kb6kb

z

10kb

M 1 2 3 1 2 3 1 2 3 1 2 3 M

Bam HI + Stu I Cla I + Fsp I dⅢ + Nde I Nco I + Xba I

(A)

(B)

圖二十三(B)、pcDNA3-NCS 經限制酵素作用後，所得之電泳分析圖

在文檔中探討登革熱二型病毒PL046之質體建構及報導基因的選擇 (頁 56-123)